This invention relates to the fractionation of lipid mixtures.
Plasma lipoproteins are spherical particles which contain varying amounts of cholesterol, triglycerides, phospholipids, and proteins. They include an outer surface composed of phospholipid, free cholesterol, and protein, and an inner core containing mostly esterified cholesterol and triglycerides. Plasma lipoproteins serve to solubilize and transport cholesterol and triglyceride in the bloodstream.
The relative proportion of protein and lipid in a plasma lipoprotein determines the density of the plasma lipoprotein, Gotto, 1988, Hosp. Pract. 23:4 (Suppl. 1). Based on their density, particle size, composition, and electrophoretic mobility, circulating lipoproteins have been categorized into four major classes. The classes are: chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Some of the characteristics of these classes are shown in Table 1.
TABLE 1 ______________________________________ CHARACTERISTICS OF PLASMA LIPOPROTEINS Diameter Density (Angstroms) (g/ml) Origin ______________________________________ Chylomicrons 750-12,000 &lt;0.95 intestine VLDL 300-700 &lt;1.006 liver LDL 180-300 1.019-1.063 catabolism of VLDL HDL 50-120 1.063-1.21 liver & intestine ______________________________________
The major classes of lipoproteins can be further divided into subclasses. LDL includes at least 7 subclasses, as is described in McNamara, 1990, AACC Lipids and Lipoproteins Division Newsletter 4:1, hereby incorporated by reference, Krauss et al., 1982, J. Lipid Res. 23:97, hereby incorporated by reference, and McNamara et al., 1987, Arteriosclerosis 7:483, hereby incorporated by reference. HDL includes at least two subclasses, HDL.sub.2 and HDL.sub.3, as is described in Whitaker, 1986, Clin. Chem. 32:1274, hereby incorporated by reference and Anderson, 1977, Biochim. Biophys. Acta 493:55, hereby incorporated by reference.
The major risk factors for coronary heart disease are hypercholesterolemia, cigarette smoking, hypertension, diabetes, obesity, and male sex. Although hypercholesterolemia in general is the most prominent of these risk factors, numerous clinical studies have shown that the different lipoprotein classes have very distinct and varied effects on heart disease, Crouse et al., 1985, J. Lipid Res., 22:566-572; Kannel et al., 1979, Ann. Intern. Med., 90:85-91; Kannel et al., 1984, Circulation 70:157A-205A. In fact the presence of HDL provides a protective effect against coronary heart disease, and therefore relatively low HDL-cholesterol levels may be indicative of greater risk, Miller et al., 1975, Lancet 16:23; Castelli et al., 1977, Circulation 55:767; Gordon et al., 1977, Am. J. Med. 62:707; Heis et al., 1980 Circulation 62:116 (Suppl. 4).
Recognition of the importance of HDL cholesterol as a strong inverse risk factor for coronary artery disease has led to substantial demand for an HDL cholesterol assay suitable for clinical and research use. Various methods, including ultracentrifugation, electrophoresis, and specific precipitation, Havel et al., 1955, J. Clin. Invest. 34:1345, have been used to separate various classes of lipoproteins.
Precipitation-based methods have been used widely for routine quantitation of lipoproteins. Several precipitation-based methods have been described in the recent literature. Most of these methods are based on earlier work by Burstein and colleagues (reviewed in Burstein, M., and Scholnick, J. R., Lipoprotein-polyanion-metal interactions, In, Advances in Lipid Research 11, R. Paoletti and D. Kritchevsky, Eds., Academic Press, New York, N.Y. 1973, pp. 67-108). In these methods, the fractionation of lipoproteins in a solution, e.g., serum, is accomplished primarily by selective precipitation followed by centrifugation. The supernatant is then used to determine the cholesterol content remaining, or, by difference, the cholesterol content of that fraction which has been specifically removed from the supernatant. For example, low density lipoprotein (LDL) cholesterol, can be determined in this manner by specifically precipitating only LDL using heparin in citrate buffer at pH 5.04. Others have reported using polyvinyl sulfate or polyethylene glycol to effect specific precipitation of LDL and VLDL. In each case, centrifugation is necessary before measuring the cholesterol content.
Similarly, the measurement of HDL-cholesterol is usually a two-step process in which HDL is first separated from the other apoB-containing plasma lipoproteins, and the cholesterol content of the HDL-containing fraction measured. The most commonly employed methods are based on those developed by Burstein and his colleagues (Burstein et al., 1988, in Clarkson TB, Kritchevsky D, Pollak OJ (eds): Monographs on Atherosclerosis, New York, Karger) in which the apoB-containing lipoproteins are removed by precipitation with a polyanion in combination with a divalent cation, Bachorik et al., 1986, Methods Enzymol. 129:78. The two most commonly used precipitants are sodium phosphotungstate-MgCl.sub.2 and dextran sulfate (m.wt. 50,000)-MgCl.sub.2, Warnick et al., 1982, Clin. Chem. 23:1379. Other precipitants used include heparin sulfate-MnCl.sub.2 and heparin sulfate-calcium carbonate. In these assays the precipitating reagent is added to an aliquot of serum or plasma. A heavy white precipitate forms immediately, and the mixture is allowed to stand until precipitation is complete. The precipitate is then sedimented by centrifugation, and an aliquot of the clear supernatant is removed for cholesterol analysis.
The first method used extensively in major population studies employed heparin and Mn.sup.++ to precipitate VLDL and LDL, allowing HDL to be measured in terms of the amount of cholesterol remaining in the supernatant solution, Burstein et al., 1960, Clin. Chem. Acta 5:609, Frederickson et al., 1968, J. Clin. Invest. 47:2446-2457, Manual of Laboratory Operations, Lipid Research Clinics Program, Lipid and Lipoprotein Analysis, I. National Heart and Lung Institute. DHEW Publications No. (NIH) 75-628, 1974. This method has been extensively studied, Bachorik et al., 1976, Clin. Chem. 22:1828-1834, Warnick et al., 1978a, J. Lipid Res. 19:65-76, and modifications have been described, Warnick et al., 1978a, supra; Warnick et al., 1978b, Clin. Chem. 24:900.